Genome-Wide Comprehensive Identification and In Silico Characterization of Lectin Receptor-Like Kinase Gene Family in Barley (Hordeum vulgare L.)

Lectin receptor-like kinases (LecRLKs) are a significant subgroup of the receptor-like kinases (RLKs) protein family. They play crucial roles in plant growth, development, immune responses, signal transduction, and stress tolerance. However, the genome-wide identification and characterization of LecRLK genes and their regulatory elements have not been explored in a major cereal crop, barley (Hordeum vulgare L.). Therefore, in this study, integrated bioinformatics tools were used to identify and characterize the LecRLK gene family in barley. Based on the phylogenetic tree and domain organization, a total of 113 LecRLK genes were identified in the barley genome (referred to as HvlecRLK) corresponding to the LecRLK genes of Arabidopsis thaliana. These putative HvlecRLK genes were classified into three groups: 62 G-type LecRLKs, 1 C-type LecRLK, and 50 L-type LecRLKs. They were unevenly distributed across eight chromosomes, including one unknown chromosome, and were predominantly located in the plasma membrane (G-type HvlecRLK (96.8%), C-type HvlecRLK (100%), and L-type HvlecRLK (98%)). An analysis of motif composition and exon-intron configuration revealed remarkable homogeneity with the members of AtlecRLK. Notably, most of the HvlecRLKs (27 G-type, 43 L-type) have no intron, suggesting their rapid functionality. The Ka/Ks and syntenic analysis demonstrated that HvlecRLK gene pairs evolved through purifying selection and gene duplication was the major factor for the expansion of the HvlecRLK gene family. Exploration of gene ontology (GO) enrichment indicated that the identified HvlecRLK genes are associated with various cellular processes, metabolic pathways, defense mechanisms, kinase activity, catalytic activity, ion binding, and other essential pathways. The regulatory network analysis identified 29 transcription factor families (TFFs), with seven major TFFs including bZIP, C2H2, ERF, MIKC_MADS, MYB, NAC, and WRKY participating in the regulation of HvlecRLK gene functions. Most notably, eight TFFs were found to be linked to the promoter region of both L-type HvleckRLK64 and HvleckRLK86. The promoter cis-acting regulatory element (CARE) analysis of barley identified a total of 75 CARE motifs responsive to light responsiveness (LR), tissue-specific (TS), hormone responsiveness (HR), and stress responsiveness (SR). The maximum number of CAREs was identified in HvleckRLK11 (25 for LR), HvleckRLK69 (17 for TS), and HvleckRLK80 (12 for HR). Additionally, HvleckRLK14, HvleckRLK16, HvleckRLK33, HvleckRLK50, HvleckRLK52, HvleckRLK56, and HvleckRLK110 were predicted to exhibit higher responses in stress conditions. In addition, 46 putative miRNAs were predicted to target 81 HvlecRLK genes and HvlecRLK13 was the most targeted gene by 8 different miRNAs. Protein-protein interaction analysis demonstrated higher functional similarities of 63 HvlecRLKs with 7 Arabidopsis STRING proteins. Our overall findings provide valuable information on the LecRLK gene family which might pave the way to advanced research on the functional mechanism of the candidate genes as well as to develop new barley cultivars in breeding programs.


Introduction
Te physiological developments of plants face constant threats from pathogenic organisms and environmental stresses.Plants have evolved mechanisms to identify pathogens through cell-surface receptors which contribute to their innate immunity and protect themselves from invading pathogens [1,2].Pattern recognition receptors (PRRs) are a crucial component of plant immunity, localized in the cell membrane where they serve as the frst line of defense by initiating early immune response [3].PRRs form complexes with other molecules, allowing them to recognize microbial molecules like pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) or damage-associated molecular patterns (DAMPs), initiating signal transduction cascades [4][5][6][7].As a result, PRRs play a pivotal role in sensing PAMPs and triggering immune responses.Plant PRRs can be categorized into two main types: receptor-like kinases (RLKs), which possess an intracellular kinase domain, and receptorlike proteins (RLPs), which lack a known intracellular signaling domain [4].
Te interaction between plants and various environmental conditions involves numerous signal recognition and transduction pathways, including the RLK superfamily, a large group of cell-surface receptors dominantly localized in the cell membrane [8].RLKs play a vital role in receiving and transmitting numerous signals and regulating various activities, such as disease resistance, self-incompatibility, hormonal sensing, and plant development [9,10].Typically, RLKs consist of three main parts: an extracellular N-terminal ligand-binding domain for signal reception, an intermediate transmembrane region for anchoring the protein in the membrane, and an intracellular C-terminal kinase domain responsible for initiating plant immunity [8,10,11].RLKs can be classifed into 17 subgroups based on the variability of the extracellular domain [12,13].In higher plants, these receptors were frst identifed in maize, and subsequently, numerous RLKs were found in over 20 plant species [14].
Lectin receptor-like kinases (LecRLKs) are characterized by the presence of an extracellular lectin domain at the Nterminus [15,16].Te diverse lectin domain at the Nterminus allows lecRLKs to recognize environmental stimuli, while the intracellular kinase domain at the Cterminus phosphorylates downstream proteins to transmit signals [15,17].Depending on the type of lectin domain, LecRLKs are further classifed into 3 subfamilies: (i) L-type, (ii) G-type, and (iii) C-type LecRLK [10].Te L-type (legume-like) LecRLKs are identifed by their lectin-legB domain and/or a protein kinase domain, mainly found in legumes [18][19][20].Despite having a β-sandwich fold structure, these proteins are soluble and exhibit glucose/mannose-binding afnity.L-type LecRLKs are found on cell membranes and have a conserved hydrophobic cavity for binding with hydrophobic ligands [21].Additionally, they play an important role in various physiological functions, including pollen development and pathogen resistance [22][23][24].G-type LecRLKs are mainly Galanthus nivalisagglutinin-related lectins which were previously named B-type LecRLKs as they have similarities in their extracellular domains with bulb lectin proteins.Having an Slocus region participating in self-incompatibility reactions, G-type LecRLKs are also known as S-domain RLKs [20,25,26].Many G-type LecRLKs contain a plasminogen apple nematode (PAN) domain and an epidermal growth factor (EGF) domain [27].Te EGF motif is cysteine-rich, likely contributing to the formation of disulfde bonds, while the PAN motif is associated with protein-protein and protein-carbohydrate interactions [28].G-type LecRLKs, such as Pi-d2 in rice, have been shown to confer resistance to the fungus Magnaporthe grisea [29] and also exhibit resistance against dark-induced leaf senescence, bacteria, and insects [30][31][32].C-type LecRLKs are a subfamily of calciumdependent RLKs which are predominantly found in mammals rather than plants [33].Tis subfamily is the smallest among plant LecRLKs, with only a single C-type lectin protein identifed in the genomes of rice and Arabidopsis (Arabidopsis thaliana) [27] and two in soybean (Glycine max) [34] and wheat (Triticum aestivum) [35].Although L-type and G-type lectin kinases are plant-specifc [10,22,36], C-type lectin kinases have been identifed in Hydra vulgaris where they are involved in immune response [37].
Barley (H.vulgare L.) is a diploid plant with 14 chromosomes and a large genome of 5.1 gigabases (Gb).It is one of the oldest domesticated cereal crops globally and holds signifcant economic value.Generally, barely is commonly used for human diets, livestock feed, and as a raw material in 2 Genetics Research the malting and brewing industries [51,52].It ranked as the fourth most abundant cereal crop in terms of cultivated area and yield (FAO: https://faosta.fao.org).Additionally, barley is one of the most stress-resistant crops, such as salt, cold, and soil infertility stress, having modulated genetic sequence organizations against biotic and abiotic stress [53].Bioinformatics analysis tools have signifcantly promoted the identifcation and in silico characterization of genes which have been developing new features day by day.Nevertheless, few bioinformatics analyses were reported on LecRLKs in various plant species, and no genome-wide identifcation and functional analysis of LecRLKs have been carried out in H. vulgare, a major economically important crop species.In this study, we comprehensively identifed LecRLK genes in barley (H.vulgare) across the genome using integrated bioinformatics approaches.We further analyzed their phylogenetic relationships, gene structures, conserved domain, motifs, chromosomal distribution, subcellular localization, gene ontology, transcription factors, and cis-regulatory elements in the promoter region.Tis study will serve as a foundational resource for in-depth studies on the functions and responses of LecRLKs to environmental stresses.

Determination of Physiochemical Properties of Barley
LecRLK Genes.Te primary transcript, gene length, chromosomal location, and open reading frame (ORF) of the identifed LecRLK genes were retrieved from the H. vulgare genome database in Phytozome.Furthermore, the basic physiochemical properties of proteins encoded by the LecRLK gene in barely, including length, molecular weight, and isoelectric points (pI), of predicted proteins, were analyzed by the online tools ExPASy (https://web.expasy.org/protparam/) [58].

Phylogenetic Relationship of LecRLK Proteins in Barley and Arabidopsis. Te protein sequences encoded by the
LecRLK gene in barely (H.vulgare) and Arabidopsis (A. thaliana) retrieved from Phytozome v13 (https:// phytozome.jgi.doe.gov/pz/portal.html/)were used to conduct the phylogenetic tree analysis.We imported all LecRLK protein sequences using MEGA 11.0 software [59] and performed multiple sequence alignments using the Clustal-W method [60] with the default parameters and 1000 bootstrap values.Finally, the phylogenetic tree was constructed using the neighbor-joining method [61] and evolutionary distances were calculated using the Equal Input method [62].Te constructed phylogenetic tree was then presented using iTOL v6.74 (https://itol.embl.de/)[63].

Gene Structure Analysis of LecRLKs in Barley.
To analyze the gene structure including exon-intron organization of predicted HvLecRLKs, CDS and genomic DNA sequences in FASTA format were obtained from Phytozome v13 (S2 Data and S3 Data).Te predicted HvLecRLK gene structure was analyzed by an online software program Gene Structure Display Server GSDS2.0 (https://gsds.cbi.pku.edu.cn/)[66] based on the DNA sequences of identifed LecRLK genes compared to the Arabidopsis LecRLK genes.

Collinearity and Synteny Analysis of the LecRLK Gene
Family of Barley.Te Plant Genome Duplication Database (https://chibba.agtec.uga.edu/duplication/index/locus) was used to confrm the gene duplication in barley and Arabidopsis lecRLK genes.Furthermore, TBtools version-v1.116 was used to illustrate the collinear and syntenic gene pairs of the HvlecRLK and AtlecRLK gene families [67].
Genetics Research 3

Analysis of Chromosomal Location of LecRLK Genes in Barley.
To predict the chromosomal location of HvLecRLKs, the barley (H.vulgare) genomic information was retrieved from the Phytozome v13 database.Chromosomal locations of the LecRLK genes of barely were determined using the tools MapGene2Chromosome V2 web server (https://mg2c.iask.in/mg2c_v2.0/)[69].

Gene Ontology Analysis of LecRLK Genes in Barley.
We used the online tool Plant Transcription Factor Database (PlantTFDB, https://planttfdb.cbi.pku.edu.cn//) to carry out the gene ontology (GO) analysis to predict the relationship of identifed LecRLK genes with the group of various biological processes, cellular processes, and molecular functions [70].

Prediction of Subcellular Localization of the Identifed
LecRLK Proteins in Barley.Te subcellular locations of the identifed LecRLK proteins were predicted in the various cell organelles by an online predictor named plant subcellular localization integrative predictor (PSI) (https://bis.zju.edu.cn/psi/) [71].

Analysis of cis-Acting Regulatory Elements (CAREs) of
HvLecRLK Gene Promoters.Te cis-acting regulatory elements (CAREs) associated with various stress responses were predicted in the 1.5 kb upstream regions of the identifed LecRLK genes by using a portal prediction tool with the Signal Scan search program in the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [73].Furthermore, predicted CAREs were divided into four classes based on their functional regulatory roles: light-responsive (LR), tissue-specifc (TS), hormoneresponsive (HR), and stress-responsive (SR).

Protein-Protein Interaction Network Prediction of
HvlecRLKs.We predicted the protein-protein interaction (PPI) network of HvlecRLKs using STRING version-11.0 (https://string-db.org/cgi/)database based on homologous protein from Arabidopsis.For PPI network analysis, STRING tool parameters were used as follows: (i) full STRING network was used as network type, (ii) the meaning of network edge evidence, (iii) interaction score was set to 0.4 (medium confdence parameter), and (iv) maximum number of interaction display is <10.  1.

Results and Discussion
In G-type HvlecRLKs, ORF length ranged from 927 bp (HvleckRLK38) to 2736 bp (HvleckRLK34), encoding potential amino acid length of 309 aa and 912 aa, respectively.Te genomic length of G-type HvLecRLKs varied from 2559 bp (HvleckRLK12) to 225550 bp (HvleckRLK16) and the molecular weight ranged from 32.4 kDa (HvleckRLK38) to 100.16 kDa (HvleckRLK34).Notably, G-type HvlecRLKs exhibited both acidic and basic properties based on their pI values.Te highest pI value was observed for HvleckRLK56 (8.8; indicating basic properties), whereas the lowest pI value was observed for HvleckRLK38 (5.31; indicating acidic properties).
C-type HvlecRLKs (HvleckRLK63) displayed an ORF length of 1845 bp encoding a potential amino acid length of 615 aa.Te genomic length and the molecular weight of the corresponding protein were 4182 bp and 67.7 kDa, respectively.C-type HvlecRLK was characterized by higher basic properties with a pI value of 9.34.Among L-type HvlecRLKs, the ORF length ranged from 1215 bp (HvleckRLK67) to 2607 bp (HvleckRLK81), encoding proteins with lengths 405 aa and 869 aa.Te genomic length of L-type HvlecRLK genes varied between 1743 bp (HvleckRLK82) and 500635 bp (HvleckRLK73).Te molecular weight ranged from 41.26 kDa (HvleckRLK67) to 95.08 kDa (HvleckRLK81).Te pI value of L-type HvLecRLK varied from 5.4 (HvleckRLK86 and HvleckRLK88) to 9.14 (HvleckRLK70).
Notably, AtleckRLK13, AtleckRLK14, AtleckRLK11, AtleckRLK15, AtleckRLK8, AtleckRLK9, and AtleckRLK10 were found to enhance H 2 O 2 (hydrogen peroxide) and cell death in response to a pathogenic bacteria like Pseudomonas syringae and pathogenic oomycetes Phytophthora infestans and Phytophthora capsici [76].Correspondingly, the HvlecRLK proteins exhibit a high activation level in response to pathogenic resistance.Additionally, AtLecRK-VI.2 (AT5G01540) was found to induce resistance against Pectobacterium carotovorum and Pseudomonas syringae [77,78] while AtLecRK-IV.3 (AT4G02410) was found to induce resistance against Botrytis cinerea [79].Several AtLecRKs such as AtLecRK-VI.2 (AT5G01540) and AtLecRK-V.5 (AT3G59700) were indeed identifed to be involved in hormone signaling (ABA) as well as stomatal immunity [77].Te majority of sequences from A. thaliana and H. vulgare are diferent, with only a total of 19 HvLecRLKs clustered with 15 AtlecRLKs revealing the distinct evolutionary functions of HvLecRLKs.A similar trend was previously identifed in Taxodium "Zhongshanshan" and other herbaceous as well as many woody plants [15,39].Moreover, LecRLKs in various woody plants formed separate clades from each other.Tus, it might be concluded that there are signifcant diferences between the LecRLK sequences among various species.

Conserved Motif Analysis of LecRLK Proteins in Barley.
Te motifs are very short active sites of enzymes facilitating the mechanism of protein folding [83].To explore conserved motifs in HvlecRLKs, the MEME program was used and identifed 20 conserved motifs distributed among G-type, Ctype, and L-type LecRLKs in barley, ranging from 04 to 20 motifs (Figure 3).In G-type HvlecRLK, 15 of them displayed the maximum number of motifs (20 motifs) indicating higher similarity with AT4G21380 (20 motifs) and were assumed to perform alike.However, the lowest number of motifs was identifed in HvleckRLK38 (04 motifs).C-type LecRLK HvleckRLK63 featured 20 motifs that were similar to the paralog AtleckRLK7.In L-type HvLecRLKs, 20 conserved motifs were predicted in 14 HvLecRLKs each while HvleckRLK67 contained only 4 conserved motifs.Ltype AtleckRLK10 and AtleckRLK9 had 18 motifs that exhibited higher conservation with HvleckRLK66, HvleckRLK68, and HvleckRLK96 each having 18 conserved motifs.Tis variation in motif numbers may contribute to the functional assortment between barley (H.vulgare) and Arabidopsis (A. thaliana).Similar motif patterns have been found in CslecRLKs of cucumber (C.sativus) and Cerasus humilis showing distinct motif features related to the variations in their protein sequences.In total, 10 conserved motifs were observed in CslecRLKs ranging from 4 to 10 in each protein and 14 conserved motifs in Cerasus humilis [84,85].Motifs 1 to 5 were predominantly identifed in Ltype CsLecRLK, whereas motif 1, motif 2, motif 6, and motif 8 were frequently observed in G-type CsLecRLK protein [84].Te variations in motif organizations indicated the functional diversity of the associated proteins.

Gene Structure Analysis of LecRLK Genes in Barley.
Evaluation of HvlecRLK gene structures revealed the exonintron confguration of the G-type, C-type, and L-type HvlecRLK genes which displayed higher conservation compared to the corresponding reference AtlecRLK genes (Figure 4).In this study, we observed that 61 Te gene structure analyses revealed that the average number of intron per HvlecRLKs was 1.5, signifcantly lower than that in cucumber genes (4.39 introns per gene) [86].A similar phenomenon has been observed in other plants.For instance, most LecRLK genes in soybeans (G.max) contained either one intron or none at all [34].Previous investigations also identifed introns in only a few LecRLK genes in Arabidopsis (A. thaliana) and rice (O.sativa).For example, out of the 75 LecRLK genes in Arabidopsis (A. thaliana) and 173 LecRLK genes in rice (O.sativa), only fve and eight genes contained intron, respectively [27].Gene structure analysis revealed the divergence of G-type, C-type, and L-type HvlecRLK genes.For instance, there are mainly 8 gene structure groups according to the number of introns (0 to 7 introns).However, in GmlecRLKs of G. max, four gene structure groups were identifed containing 3 introns, six introns, seven introns, and no introns in their coding sequences [34].It has been previously demonstrated that introns play a pivotal role in cellular processes as well as Genetics Research plant developmental processes by regulating gene expression or alternative splicing [87].Notably, most of the L-type LecRLKs in both H. vulgare and G. max have no intron demonstrating that they are more conserved and showed less divergence in structure [34].Te compact gene structure is expected to enhance transcriptomic gene expression by inhibiting variable splicing and reducing energy consumption, particularly for genes responding to various environmental stresses.

Ka/Ks Analysis of HvlecRLK Gene
Family.Te values of Ka (nonsynonymous substitutions) and Ks (synonymous substitutions) and Ka/Ks ratios were analyzed to determine the selection pressure and evolutionary history of lecRLKs in barley (H.vulgare) (Figure 5).In total, 28 homologous pairs of HvlecRLKs were determined.During the evolutionary period, genes evolved from various selection pressures, such as purifying selection, natural selection, and positive selection.Our investigation determined the Ka/Ks ratios for   [65].Each color represents diferent motifs within the predicted protein domains.(HvleckRLK75-HvleckRLK109) to 0.86 (HvleckRLK38-HvleckRLK46) indicating the evolution through purifying selection of these paired genes.Te Ka/Ks ratios of all duplicated lecRLK genes in soybean (G.max) were less than 0.5, also suggesting evolution through purifying selection [34].However, in cucumber (C.sativus) [84] and peanut (Arachis hypogaea) [88], both positive and purifying selections were determined in duplicated CslecRLK and AhlecRLK genes.Furthermore, we analyzed the divergence period of duplicated HvlecRLKs ranging from 1.25E-16 (HvleckRLK11-HvleckRLK12) to 1.09E-15 (HvleckRLK6-HvleckRLK44) with an average duplication time of 1.74E-15 MYA, demonstrating the recent gene duplication events of HvlecRLKs in barley (H.vulgare).Similar fndings were also observed in AhRLK genes of Arachis hypogaea in which the divergence period ranged from 0 to 2 MYA illustrating their evolution through recent gene duplication events [88].It might be concluded that HvlecRLKs underwent duplication before their existence with several potential functions.

Collinearity and Synteny Analysis of the LecRLK Gene
Family in Barley.To determine the evolutionary relationship between the lecRLK gene family of barley and Arabidopsis, a comprehensive collinearity analysis was conducted (Figure 6(a)).Collinearity, a particular form of synteny, requires specifc gene order [89].Tis investigation showed that 34 collinear pairs were identifed within HvlecRLK genes, with the highest number of collinear genes found in chromosome 2 (12) followed by chromosome 7 (09), chromosome 3 (08), chromosome 5 (07), chromosome 6 (06), and chromosome 1 (05).Furthermore, two collinear genes were identifed in an unknown chromosome and the least number was observed in chromosome 4 (01).Tese collinear HvlecRLK gene pairs were involved in lineagespecifc expansion over evolution [90].Moreover, synteny analysis was also conducted to reveal the expansion mechanism and evolutionary relationship of the lecRLK gene family between barley and Arabidopsis genome (Figure 6(b)).In total, 7 syntenic gene pairs were identifed showing higher homology with AtlecRLKs.Te syntenic analysis was also previously performed in cucumber lecRLK    12 Genetics Research genes identifying higher homology between CslecRLKs and AtlecRLK [84].Tis study suggests that the HvlecRLK genes were highly conserved having similar ancestors with which performed similar functions.

Analysis of Chromosomal Location of LecRLK Genes in
Barley.We investigated the chromosomal locations of barley LecRLKs to understand the genomic distribution of the predicted genes (Figure 7).Tis study revealed that mapped G-type, C-type, and L-type HvlecRLK genes were located on 8 individual chromosomes including an unknown chromosome (ChrUn) within 770 Mb in the entire genome of barley (H.vulgare) (Figure 5).Te number of HvlecRLKs on each chromosome ranged from 3 to 31, with Chr2H containing the highest number of HvlecRLKs (31) while chr4H had only 3 HvlecRLKs.Four HvlecRLKs were identifed in an unknown chromosome.All 62 G-type HvlecRLK genes were distributed across 8 independent chromosomes, with 5, 20, 9, 01, 6, 6, and 13 HvlecRLKs in Chr1H to Chr7H, respectively.Two G-type HvlecRLKs (HvleckRLK1, HvleckRLK2) were found on ChrUn.A single C-type HvlecRLK gene was located on Chr3H (HvleckRLK63).Among the 50 L-type HvlecRLKs, number 5, 11, 6, 2, 8, 8, and 8 HvlecRLKs were unevenly distributed on Chr1H-Chr7H, respectively, while HvleckRLK64 and HvleckRLK65 were located on an unknown chromosone (designated as ChrUn).Our fnding showed similarity to previous investigations on LecRLKs of cucumber (C.sativus) [42], potato (S. tuberosum) [41], and soybean (G.max) [34] in which LecRLK genes were unevenly scattered on a total of 7, 12, and 19 chromosomes, respectively.In cucumber, the highest number of CslecRLKs (12) was located on chromosome 3 while in potato, the largest number of StlecRLks (20) was identifed on chromosome 7 [41,42].However, In G. max, chromosome 4 and chromosome 18 contained only G-type and L-type GmlecRLKs, separately, and 17 chromosomes consisted of both G-type and L-type GmlecRLKs.
Additionally, the largest number of GmlecRLks was located on chromosome 6, chromosome 12, and chromosome 13 [34].Furthermore, ChLecRLK genes of C. humilis were found to be unevenly distributed through eight chromosomes consisting of the majority of ChLecRLK genes (56) on chromosome 3 and lowest on chromosome 8 (3) [85].

Prediction of Subcellular Localization of the Identifed
LecRLK Proteins in Barley.Te study of subcellular localization revealed the cellular appearance of the reported proteins.In this investigation, the majority of HvlecRLK proteins were predicted in the plasma membrane (G-type HvlecRLK is 96.77%,C-type HvlecRLK is 100%, and L-type HvlecRLK is 98%) followed by extracellular region (G-type HvlecRLK is 24.19%,C-type HvlecRLK is 0%, and L-type HvlecRLK is 2%) and chloroplast (G-type HvlecRLK is 4.83%, C-type HvlecRLK is 0%, and L-type HvlecRLK is 18%) (Figure 9).Te LecRLK proteins located in the plasma membrane play roles in connecting the cell wall and membrane, facilitating transmembrane movements, and ultimately regulating plant responses to pathogen attacks

Classification
Figure 8: Te gene ontology (GO) terms correspond to HvLecRLK genes.Te predicted GO terms corresponding to the reported HvlecRLK genes are presented for biological processes, cellular components, and molecular functions whether the genes are associated or not.Te p value corresponding to the GO terms is shown in the histogram, using -log10 (p value).
14 Genetics Research [84].However, we observed that one G-type HvlecRLK, HvleckRLK2, appeared in the nuclear region and one L-type HvlecRLK, HvleckRLK91, appeared in the cytoplasmic region.It is worth noting that C-type HvlecRLK was also found in the nucleus and mitochondria.Previous studies have shown that LecRLK proteins present in mitochondria play a crucial role in plant growth and stress response mechanisms [96].Te majority of TzlecRLKs proteins (71.7%) in Taxodium "Zhongshanshan" and StlecRLKs proteins (77%) in S. lycopersicum were located in the plasma membrane which also support our fnding subcellular localization analysis [15,41].Te remaining LecRLKs are present in other cellular loci such as mitochondria, chloroplast, vacuole, and nucleus.According to the result, we can speculate that the HvlecRLks are not limited to the cell membrane but the other cellular organelles.Tus, the HvlecRLKs found in several loci might be expressed in the whole cell system.
Te ERF TFF was recognized as one of the largest families which have been previously determined [104].ERF family members play a crucial role in plant hormonal response under stressful conditions including response to abscisic acid and ethylene to activate stress-responsive genes and enhance salt and drought tolerance response in tomato [105,106].Te WRKY family is known for its role in boosting defense mechanisms against pathogens in various plant species [107,108].Both bZIP and TFF control gene expression for plant development under abiotic stress [109,110].Te MIKC-MADS TFF includes members with diverse functions in vegetative and reproductive phases, regulating genes associated with pollen, fower, endosperms, and root development [111].Another important TFF C2H2 having a fnger-like structure can bind zinc ions and respond to environmental stimuli [112].On the other hand, MYB TFF is involved in cell identity, seed, and fower development, defense and stress responses, and primary and secondary metabolism regulation [113][114][115].In plants, Dof TFF (DNA-binding one fnger) plays a pivotal role in transcriptional regulation due to its dual functionality in binding to both DNA and proteins [116,117].Furthermore, it contributes to seed maturation and germination, plant hormone regulation, and resistance response to various stresses [116][117][118].Te enrichment of TFF might be a major source of functional diversity in plant genomes [119].Te interaction between TFs and the identifed genes in barley represents an extensive variability of gene expression pattern which can be explored thoroughly by further investigation in wet lab experiments.

Analysis of cis-Acting Regulatory Elements (CAREs) of
HvlecRLK Gene Promoters.Te cis-acting regulatory elements (CAREs) mainly consist of short DNA motifs (5-20 bp) located in the promoter region of the target gene.Te CAREs predicted in the gene promoter provide valuable information about their roles in plant growth, development, and stress response [120].Our analysis identifed a total of 12648 cis-elements belonging to 75 CARE motifs including 36 diferent types of CARE motifs associated with lightresponsive (LR) functions, 21 tissue-specifc (TS) functions, 13 hormone-responsive (HR) functions, and 5 stressresponsive (SR) functions in the promoter regions of HvlecRLKs (Figure 11(a)).When comparing with all four motif categories, the highest number of cis-elements was detected in HR categories at 39.60%, followed by LR at 32.15%, TS 21.17%, and SR 7.09%.Tese cis-elements play a vital role in plant defense mechanisms and various stress responses [121][122][123].On the other hand, CARE motifs belonging to the LR categories were abundant in the HvlecRLKs promoter region which is associated with photosynthesis.Photosynthesis is an important physiological process infuenced by the light response in barley leaf tissue [124].LR motifs such as G-box (31.31%),G-Box (10.01%),Sp1 (8.73%), GT1-motif (6.49%), and TCT-motif (6.98%) were predominantly found in 101, 99, 89, 67, and 63 HvlecRLK genes, respectively (Figure 11(b)).Notably, the highest number of LR motifs was found in the regulatory region of HvleckRLK11 (25 motifs), HvleckRLK50 (24 motifs), HvleckRLK73 (24 motifs), and HvleckRLK80 (24 motifs), respectively.Previous research has also demonstrated the signifcant role of these LR motifs in the light response of various plant species [124][125][126][127].

Conclusion
In this study, we utilized the integrated bioinformatics approaches for the in silico identifcation and characterization of LecRLK genes in the barley genome (H.vulgare L.).A total of 113 LecRLK genes were identifed and phylogenetically classifed into three main categories (G-type, Ctype, and L-type HvlecRLK) which maintain a close evolutionary relationship with AtlecRLKs.Te predicted chromosomal location revealed that these HvlecRLK genes were unevenly distributed across 8 chromosomes including an unknown chromosome.Te domain, motif, and exonintron organization of HvlecRLKs demonstrated remarkable homogeneity with the corresponding gene family of Arabidopsis.Te Ka/Ks ratios and collinear and syntenic gene pairs provide insight into the evolution of HvlecRLK genes.Furthermore, the GO analysis revealed the involvement of the identifed HvlecRLk genes in several crucial biological, cellular, and molecular functions.Te subcellular localization analysis identifed the maximum protein signal in the plasma membrane indicating their involvement in the defense mechanism.Te regulatory network and subnetwork analysis determined the presence of 29 TFFs including AP2, bZIP, C2H2, Dof, ERF, MIKC_MADS, MYB, NAC, and WRKY families linked to the putative LecRLK genes of barley.Furthermore, the cis-acting element analysis demonstrated the presence of CAREs in the HvlecRLKs promoter region associated with the response to light, tissuespecifc, hormone, and stress.Te predicted TFs were expected to bind with the CAREs of HvlecRLKs boosting plant growth and development as well as LecRLK gene expression of barley (H.vulgare).Tus, the fndings might provide a strong basis for further functional investigation, characterization, and improvement of the LecRLK genes in wet lab experiments.Tis research has the potential to be valuable in breeding programs for this economically important cereal grain in the future.

Figure 1 :
Figure 1: Te phylogenetic relationship between barley and Arabidopsis LecRLK family proteins.Phylogenetic tree representing the evolutionary relationship for the G-type LecRLK, C-type LecRLK, and L-type LecRLK proteins from H. vulgare and Arabidopsis.Te phylogenetic trees were constructed using the neighbor-joining method.Diferent groups present here are indicated by diferent colors.Te red dots represent the Arabidopsis lecRLK proteins and the blue lines represent the barley lecRLK proteins.

Figure 2 :
Figure 2: Feature domain of Hordeum vulgare L. LecRLK proteins.Te conserved domains of the identifed HvlecRLK proteins were drawn by using the Pfam database [64].Te position of the identifed domain is demonstrated by diferent colored boxes including the domain name.

Figure 3 :
Figure 3: Te distribution of conserved motifs in barley LecRLK protein.Te distribution of conserved motifs of the predicted G-type, Ctype, and L-type HvlecRLK protein families is illustrated using MEME-suite (https://meme-suite.org/meme/) (a maximum of 20 motifs are displayed) [65].Each color represents diferent motifs within the predicted protein domains.

Figure 4 :Figure 5 :
Figure4: Te gene structure of barley LecRLK genes.Gene structure of the predicted G-type, C-type, and L-type LecRLK genes in H. vulgare compared to A. thaliana is illustrated using Gene Structure Display Server (GSDS 2.0, https://gsds.cbi.pku.edu.cn/index.php)[66].Gene families are categorized based on their phylogenetic relationship.For all HvlecRLK genes, black lines represent introns, green-bold lines represent exons, and red-bold lines represent 5′ and 3′ untranslated regions (UTR).Te gene structure of each HvlecRLK is displayed according to the scale mentioned at the bottom.

Figure 6 :
Figure 6: Te collinearity and syntenic relationships between barley (H.vulgare) and Arabidopsis (A. thaliana).(a) Te collinearity analysis of the LecRLK gene family in barley.Te colored rectangles represent chromosomes 1-7 with an unknown chromosome.Te collinear blocks are represented with colored lines.(b) Te synteny analysis of LecRLK genes between barley and Arabidopsis.Te colored rectangles represent chromosomes 1-7 with an unknown chromosome and the red colored lines represent the synteny blocks.

Figure 7 :
Figure 7: Te chromosomal location of HvlecRLK genes.Te chromosomal location of the predicted HvlecRLK genes is illustrated.Te chromosome number is at the top of each chromosome bar.Te scale to indicate the chromosomal length as millions of bases (Mb) is provided on the left based on the information retrieved from Phytozome v13 [54].ChrUn means the unknown chromosome.

Figure 9 :
Figure 9: A heatmap represents the subcellular localization of barley HvlecRLK protein.Subcellular localizations for the G-type, C-type, and L-type HvlecRLK proteins are shown in the heatmap.Te names of each HvlecRLK protein are displayed on the left side of the heatmap, with the terms of the respective cellular organelles displayed at the bottom.Te color intensity on the right side of the heatmap shows the presence of protein signals associated with the genes.In this study, reported proteins were analyzed in the plasma membrane, extracellular region, chloroplast, nucleus, mitochondria, and cytoplasmic region.

Figure 10 :
Figure 10: Te distribution of transcription factors on the promoter region of HvLecRLK genes.LecRLK gene-mediated subnetwork for bZIP, C2H2, ERF, MIKC_MADS, MYB, NAC, and WRKY TFs families which is expressed as heatmap.Te name of each gene is shown on the left side of the heatmap.

Figure 11 :
Figure 11: Te distribution of cis-regulatory elements in the promoter region of the identifed G-type, C-type, and L-type HvLecRLK genes.(a) Te distribution of cis-regulatory elements in the HvlecRLK promoter region is illustrated as a heatmap.Te names of each HvlecRLK gene are displayed on the left side of the heatmap.Te green, orange, red, and blue colors represent CAREs of corresponding HvLecRLKs such as light responsiveness (LR), tissue-specifc (TS), phytohormone responsiveness (HR), and stress responsiveness (SR), respectively.Te percentage (%) ratio of the numerous cis-elements from each category is presented in pie charts: (b) light-responsive; (c) tissue-specifc; (d) phytohormones-responsive; (e) stress-responsive.(f ) Te percentage (%) of HvlecRLK genes involved in four categories of cis-elements.

Figure 12 :
Figure 12: Predicted miRNAs targeted HvlecRLK genes.(a) Network illustration of predicted miRNA targeting HvlecRLK genes.Light blue rectangles represent the putative miRNAs and red oval shapes represent the targeted HvlecRLK genes.(b) Te schematic diagram represents the HvlecRLK genes targeted by miRNAs and the red color represents the putative miRNAs sites of each gene.

Figure 13 :
Figure 13: Te protein-protein interaction network of HvlecRLK proteins.Te proteins are represented at network nodes and the colored lines indicate diferent data sources.Te thicker interaction lines between proteins indicate the higher coefcient and vice versa.

Table 1 :
List of 113 LecRLK genes of barley and their basic physiochemical characterization.

Table 1 :
Continued.Te phylogenetic tree analysis revealed the evolutionary relationship between G-type, C-type, and Ltype LecRLK proteins in barley and Arabidopsis with AtlecRLK protein sequences as query sequences (Figure Te well-conserved gene structure of HvlecRLK genes with Arabidopsis (A. thaliana) suggests similar functional activity.
.95% of HvlecRLKs (70 out of 113) were intron-less.Te highest number of introns (7 introns) was identifed in HvleckRLK7, HvleckRLK25, and HvleckRLK47 belonging to the G-type LecRLK subfamily.Among the 62 G-type HvlecRLKs, 27 genes had no intron while the remaining exhibited a variable number of introns.Some members of HvlecRLK exhibited similar exon-intron organization while many had a lower number of introns compared to G-type AtlecRLK.C-type HvlecRLK carrying 4 exons and 3 introns was just one less than C-type AtlecRLK.Most L-type HvlecRLKs exhibited structural similarity to the corresponding Arabidopsis (A. thaliana) genes.Notably, 43 members had

Table 2 :
Information about abundant miRNA ID, functions, and their targeted HvlecRLK genes.